The invention relates to vaccines and in particular, live vaccines against Actinobacillus pleuropneumoniae (APP) and related bacterial pathogens. The invention is also concerned with recombinant techniques for preparing such a vaccine.
An organism known as Actinobacillus pleuropneumoniae (APP) is a gram negative coccobacillus organism that is found in the pig and causes pneumonia in the pig.
This disease is characteristically an acute necrotizing hemorrhagic bronchopneumonia, with accompanying fibrinous pleuritis (Fenwick, B. and S. Henry. 1994. Porcine pleuropneumonia. J. Am. Vet. Med. Assoc. 204:1334-1340) (Sebunya, T. N. K. and J. R. Saunders. 1983. Haemophilus pleuropneumoniae infection in swine: a review. J. Am. Vet. Med. Assoc. 182:1331-1337). Porcine pleuropneumonia is an economically devastating, severe and often fatal disease with clinical courses ranging from hyperacute to chronic infection (Fenwick, B. and S. Henry. 1994. Porcine pleuropneumonia. J. Am. Vet. Med. Assoc. 204:1334-1340) (Hunneman, W. A. 1986. Incidence, economic effects, and control of Haemophilus pleuropneumoniae infections in pigs. Vet. Quarterly 8:83-87). The existence of at least twelve antigenically distinct capsular serotypes (Perry, M. B., E. Altman, J.-R. Brisson, L. M. Beynon, and J. C. Richards. 1990. Structural characteristics of the antigenic capsular polysaccharides and lipopolysaccharides involved in the serological classification of Actinobacillus pleuropneumoniae strains. Serodiag. Immunother. Infect. Dis. 4:299-308) has made development of a cross-protective vaccine difficult. Killed whole cell bacterins provide at best serotype-specific protection (Nielsen, R. 1984. Haemophilus pleuropneumoniae serotypesxe2x80x94Cross protection experiments. Nord. Vet. Med. 36:221-234) (Nielsen, R. 1976. Pleuropneumonia of swine caused by Haemophilus pleuropneumoniae. Studies on the protection obtained by vaccination. Nord. Vet. Med. 28:337-338) (Rosendal, S., D. S. Carpenter, W. R. Mitchell, and M. R. Wilson. 1981. Vaccination against pleuropneumonia in pigs caused by Haemophilus pleuropneumoniae. Can. Vet. J. 22:34-35) (Thacker, B. J., and M. H. Mulks. 1988. Evaluation of commercial Haemophilus pleuropneumoniae vaccines. Proc. Int. Pig Vet. Soc. 10:87). In contrast, natural or experimental infection with a highly virulent serotype of A. pleuropneumoniae elicits protection against reinfection with any serotype (Nielsen, R. 1979. Haemophilus parahaemolyticus serotypes: pathogenicity and cross immunity. Nord. Vet. Med. 31:407-413) (Nielsen, R. 1984. Haemophilus pleuropneumoniae serotypesxe2x80x94Cross protection experiments. Nord. Vet. Med. 36:221-234) (Nielsen, R. 1974. Serological and immunological studies of pleuropneumonia of swine caused by Haemophilus parahaemolyticus. Acta Vet. Scand. 15:80-89). In several recent studies, attenuated strains of A. pleuropneumoniae produced by chemical nutagenesis, serial passage, or other undefined spontaneous mutation have been tested as live vaccines, with promising results (Inzana, T. J., J. Todd, and H. P. Veit. 1993. Safety, stability and efficacy of nonencapsulated mutants of Actinobacillus pleuropneumoniae for use in live vaccines. Infect. Immun. 61:1682-1686) (Paltineanu, D., R. Pambucol, E. Tirziu, and I. Scobercea. 1992. Swine infectious pleuropneumonia: Aerosol vaccination with a live attenuated vaccine. Proc. Int. Pig. Vet. Soc. 12:214) (Utrera, V., C. Pijoan, and T. Molitor. 1992. Evaluation of the immunity induced in pigs after infection with a low virulence strain of A. pleuropneumoniae serotype 1. Proc. Int. Pig. Vet. Soc. 12:213). However, the use of live vaccines in the field is problematic, particularly when the attenuating lesion in the vaccine strain has not been genetically defined. A well-defined mutation that prevents reversion to wild-type would be extremely desirable for the development of a live attenuated vaccine against Actinobacillus pleuropneumoniae. 
A variety of mutations in biosynthetic pathways are known to be attenuating in other organisms. Lesions in aro(Hoiseth S. K. and B. A. D. Stocker. 1981. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature (london). 291: 238-239) (Homchampa, P., R. A. Strugnell and B. Adler. 1992. Molecular analysis of the aroA gene of Pasteurella multocida and vaccine potential of a constructed aroA mutant. Mol. Microbiol. 6: 3585-3593) (Homchampa, P., R. A. Strugnell and B. Adler. 1994. Construction and vaccine potential of an aroA mutant of Pasteurella haemolytica. Vet. Microbiol. 42:35-44) (Karnell, A., P. D. Cam, N. Verma and A. A. Lindberg. 1993. AroD deleteion attenuates Shigella flexneri strain 2457T and makes it a safe and efficacious oral vaccine in monkeys. Vaccine 8:830-836.) (Lindberg, A. A., A. Karnell, B. A. D. Stocker, S. Katakura, H. Sweiha and F. P. Reinholt. 1988. Development of an auxotrophic oral live Shigella flexneri vaccine. Vaccine 6:146-150) (O""Callaghan, D. D. Maskell, F. Y. Lieu, C. S. F. Easmon and G. Dougan. 1988. Characterization of aromatic and purine dependent Salmonella typhimurium: attenuation, persistence and ability to induce protective immunity in BALB/c mice. Infect. Immun. 56:419-423) (Vaughan, L. M., P. R. Smith, and T. J. Foster. 1993. An aromatic-dependent mutant of the fish pathogen Aeromonas salmonicida is attenuated in fish and is effective as a live vaccine against the Salmonid disease furunculosis. Infect. Immun. 61:2172-2181), pur (O""Callaghan, D. D. Maskell, F. Y. Lieu, C. S. F. Easmon and G. Dougan. 1988. Characterization of aromatic and purine dependent Salmonella typhimurium: attenuation, persistence and ability to induce protective immunity in BALB/c mice. Infect. Immun. 56:419-423) (Sigwart, D. F., B. A. D. Stocker, and J. D. Clements. 1989. Effect of a purA mutation on the efficacy of Salmonella live vaccine vectors. Infect. Immun. 57:1858-1861), and thy (Ahmed, Z. U., M. R. Sarker, and D. A. Sack. 1990. Protection of adult rabbits and monkeys from lethal shigellosis by oral immunization with a thymine-requiring and temperature-sensitive mutant of Shigella flexneri Y. Vaccine. 8:153-158) loci, which affect the biosynthesis of aromatic amino acids, purines, and thymine, respectively, are attenuating because they eliminate the ability of the bacterium to synthesize critical compounds that are not readily available within mammalian hosts. For example, aro mutants of Salmonella and Shigella species have been shown to be attenuated in their natural hosts (Hoiseth S. K. and B. A. D. Stocker. 1981. Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines. Nature (london). 291: 238-239) (Homchampa, P., R. A. Strugnell and B. Adler. 1992. Molecular analysis of the aroA gene of Pasteurella multocida and vaccine potential of a constructed aroA mutant. Mol. Microbiol. 6: 3585-3593) (Homchampa, P., R. A. Strugnell and B. Adler. 1994. Construction and vaccine potential of an aroA mutant of Pasteurella haemolytica. Vet. Microbiol. 42:35-44) (Karnell, A., P. D. Cam, N. Verma and A. A. Lindberg. 1993. AroD deletion attenuates Shigella flexneri strain 2457T and makes it a safe and efficacious oral vaccine in monkeys. Vaccine 8:830-836) (Lindberg, A. A., A. Karnell, B. A. D. Stocker, S. Katakura, H. Sweiha and F. P. Reinholt. 1988. Development of an auxotrophic oral live Shigella flexneri vaccine. Vaccine 6:146-150) (O""Callaghan, D. D. Maskell, F. Y. Lieu, C. S. F. Easmon and G. Dougan. 1988. Characterization of aromatic and purine dependent Salmonella typhimurium: attenuation, persistence and ability to induce protective immunity in BALB/c mice. Infect. Immun. 56:419-423). Lesions that affect the biosynthesis of LPS (Collins, L. V., S. Attridge, and J. Hackett. 1991. Mutations at rfc or pmi attenuate Salmonella typhimurium virulence for mice. Infect. Immun. 59:1079-1085) (Nnalue, N. A., and B. A. D. Stocker. 1987. Tests of the virulence and live-vaccine efficacy of auxotrophic and gale derivatives of Salmonella cholerasuis. Infect. Immun. 55:955-962) and of cyclic AMP (Kelly, S. M., B. A. Bosecker and R. Curtiss III. 1992. Characterization and protective properties of attenuated mutants of Salmonella cholerasuis. Infect. Immun. 60:4881-4890) (Tacket, C. I., D. M. Hone, R. Curtiss III, S. M. Kelly, G. Losonsky, L. Guers. A. M. Harris, R. Edelman. M. M. Levine. 1992. Comparison of the safety and immunogenicity of xcex94aroC xcex94aroD and xcex94cyaxcex94crp Salmonella typhi strains in adult volunteers. Infect. Immun. 60:536-541) have also been shown to be attenuating in Salmonella species. It is important to note that not all attenuating mutations are good vaccine candidates in different organisms because some attenuating mutations result in poor persistence and immunogenicity (O""Callaghan, D. D. Maskell, F. Y. Lieu, C. S. F. Easmon and G. Dougan. 1988. Characterization of aromatic and purine dependent Salmonella typhimurium: attenuation, persistence and ability to induce protective immunity in BALB/c mice. Infect. Immun. 56:419-423) (Sigwart, D. F., B. A. D. Stocker, and J. D. Clements. 1989. Effect of a purA mutation on the efficacy of Salmonella live vaccine vectors. Infect. Immun. 57:1858-1861).
Riboflavin (vitamin B2) , a precursor of the coenzymes flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), is essential for basic metabolism. It is synthesized by plants and by most microorganisms but not by higher animals (Bacher, A. 1991. Biosynthesis of flavins. p. 215-59. In F. Muller (ed.), Chemistry and Biochemistry of Flavins, Vol. 1. Chemical Rubber Company, Boca Raton, Fla.). Many pathogenic bacteria are apparently unable to utilize flavins from their environment and are entirely dependent on endogenous production of riboflavin (Schott, K., J. Kellerman, F. Lottspeich and A. Bacher. 1990. Riboflavin syntheses of Bacillus subtilis: purification and amino acid sequence of the xcex1-subunit. J. Biol.Chem. 265:4204-4209). Even with the ability to utilize exogenous riboflavin, there may not be enough of the vitamin present in mammalian host tissues to permit growth, particularly not in sites devoid of normal bacterial flora.
Vaccines are preparations used to prevent specific diseases in animals by inducing immunity. This is accomplished by exposing a patient to an antigen from an agent capable of causing a particular disease which, in turn, causes the immune system of the patient to produce large Quantities of antibody. The presence of the antibody in the patient""s blood protects the patient from a later attack by the disease-causing agent. Vaccines may either be composed of subunits of the agent, or the live or killed agent itself. If a live vaccine is to be used, its virulence must be attenuated in some way; otherwise, the vaccine will cause the disease it is intended to protect against. See U.S. Pat. No. 5,429,818, Col. 1.
Most current vaccines against APP are killed whole cell bacterins, that is, whole bacterial cells killed by heat treatment or formalinization, suspended in an adjuvant solution. Some alternative ways of attempting to develop vaccines against APP are the use of subunit vaccines and the use of non-encapsulated mutants.
The use of a protease lysate of the outer membrane of A. pleuropneumoniae cells as a vaccine against APP infection is described in U.S. Pat. No. 5,332,572.
The use of extracellular proteins and/or hemolysins from APP as vaccines against APP infection is described in U.S. Pat. No. 5,254,340, WO Patent No. 9409821, EP No. 595,188, CA 2045950, and EP No. 453,024.
The use of non-encapsulated mutants of APP is described in U.S. Pat. No. 5,429,818. It disclosed that the capsule of such bacteria is required for virulence. Therefore, the preparation of a mutant of APP that was a non-capsulated mutant was described as a vaccine.
A method of administering vaccines to pigs by a transthoracic intrapulmonary immunization is described in U.S. Pat. No. 5,456,914.
A vaccine for the immunization of an individual against Salmonella choleraesuis utilizing derivatives that are incapable of producing functional adenylate cyclase and/or cyclic AMP receptor protein is described in U.S. Pat. No. 5,468,485. The avirulent S. choleraesuis was made avirulent by an inactivating mutation in a cya gene and an inactivating mutation in a crp gene. Similar techniques are described in other bacteria in U.S. Pat. Nos. 5,424,065; 5,389,386; 5,387,744 and 4,888,170.
To protect animals from lung disease, it is needed to achieve a sufficiently high level of antibodies, particularly IgA antibodies, in the lungs to prevent adherence of invading microorganisms to mucosal surfaces and neutralize potentially damaging virulence factors. Antibodies in the patient""s serum or at the mucosal surfaces can be important to protection. One of the reasons for using a live vaccine instead of a killed whole cell bacterin is that a live vaccine, given intranasally or orally, can induce specific local secretory antibody in the secretions that cover mucosal surfaces. This local antibody is often quite helpful for protection against diseases that infect at or through mucosal surfaces.
None of the patents pertain to a recombinant technique for a relatively convenient method for obtaining genetically defined mutants for use in a vaccine against APP.
It is believed that a mutation in a critical biosynthetic pathway which limits growth in vivo but does not otherwise alter expression of important antigens such as capsular polysaccharide, lipopolysaccharide and extracellular toxins, could produce an attenuated vaccine strain capable of inducing cross-protective immunity against A. pleuropneumoniae. 
It is believed that riboflavin biosynthesis would be essential for survival of A. pleuropneumoniae in vivo, and that mutations in the riboflavin biosynthetic pathway would be attenuating due to the scarcity of riboflavin present on the mucosal surfaces of the respiratory tract.
It is an object of the present invention to describe the use of mutations in the riboflavin biosynthetic pathway to construct attenuated strains of pathogenic bacteria for use as live vaccines, with a riboflavin-requiring mutant of APP used as a specific example.
It is an object of the present invention to describe a live vaccine against APP utilizing a riboflavin mutation in the APP genome.
Described is a live vaccine against bacterial pathogens comprising a recombinant riboflavin-requiring mutant having a mutation that inactivates riboflavin biosynthesis therein. In particular, this includes bacterial pathogens in the family Pasteurellaceae, which include animal pathogens as Actinobacillus pleuropneumoniae, Actinobacillus suis, Haemophilus parasuis, Pasteurella haemolytica and Pasteurella multocida, as well as human pathogens Haemophilus influenzae and Haemophilus ducreyi. 
Also described is a live vaccine against Actinobacillus pleuropneumoniae (APP) comprising a recombinant APP having an attenuating inactivating mutation therein.